WO2011073883A1 - Pichia pastoris déficiente en protéase endogène sécrétée - Google Patents

Pichia pastoris déficiente en protéase endogène sécrétée Download PDF

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WO2011073883A1
WO2011073883A1 PCT/IB2010/055777 IB2010055777W WO2011073883A1 WO 2011073883 A1 WO2011073883 A1 WO 2011073883A1 IB 2010055777 W IB2010055777 W IB 2010055777W WO 2011073883 A1 WO2011073883 A1 WO 2011073883A1
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pichia pastoris
sub2
gene
subtilisin
cell
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PCT/IB2010/055777
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Michel Monod
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Centre Hospitalier Universitaire Vaudois (Chuv)
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Priority to EP10814699A priority Critical patent/EP2513301A1/fr
Priority to US13/515,577 priority patent/US8703444B2/en
Publication of WO2011073883A1 publication Critical patent/WO2011073883A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
    • C12N9/60—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi from yeast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present invention relates to micro-organisms and to methods of producing proteins. More specifically, the inventions relates to a host cell useful for the expression of heterologous proteins in which the host cell, Pichia pastoris, has been genetically modified.
  • the yeasts have received broad attention in recent years, and offer some advantages of the prokaryotic system, such as simple genetic manipulation and rapid growth.
  • the yeast organisms are able to make post-translational protein modifications typical of eukaryotic cells.
  • those employed as expression systems of interest is Pichia pastoris, which has received wide acceptance for the production of biopharmaceuticals, since it is capable of doing some post-translational process, such as glycosylation, so very similar to mammals.
  • Pichia pastoris One of the most important features of Pichia pastoris is its ability to grow in culture medium containing methanol as the sole source of carbon.
  • the first step in the use of methanol is the oxidation of this, leading to the formation of formaldehyde and hydrogen peroxide, a reaction catalyzed by the enzyme alcohol oxidase.
  • Pichia pastoris has two genes that encode for alcohol oxidase, AOXl and AOX2, but only the former is heavily regulated by methanol while the second is expressed in low quantities.
  • AOXl alcohol oxidase enzyme 1
  • the control of AOXl gene expression occurs at transcriptional level.
  • the promoter of the gene AOXl has been widely used for the construction of expression vectors of Pichia pastoris for being strong and highly adjustable, which reduces the possibility of toxic proteins harming the cell growth. These vectors are integrative type, allowing the stabilization of the exogenous gene message in the genome of Pichia pastoris.
  • Pichia pastoris secretes very low levels of native proteins. More interesting, there have been no reports of extracellular proteases in Pichia pastoris and studies suggest that most of the proteases active in Pichia pastoris are intracellular and not secreted (Jarnaa Sinha et al., Biotechnology and Bioengineering, 89: 102-1 12, 2005).
  • Still another important feature is that the expression of heterologous proteins or polypeptides in Pichia pastoris has shown high levels of production when grown at high cell densities in simple defined medium. Indeed, the strong promoter, coupled with the high cell density fermentation, has allowed production of heterologous proteins or polypeptides at high concentration.
  • vacuolar proteases are required for several developmental transitions in the life cycle of yeast cells (e.g., sporulation), they are dispensible for vegetative growth.
  • the majority of vacuolar proteases are synthesized and transported through the secretory pathway as enzymatically inactive zymogens (Klionsky et al., Microbiol. Rev. 54:266-292, 1990; Raymond et al., Int. Rev. Cytol. 139:59-120, 1992). They are proteolytically activated by the combined action of proteinase A, the product of the PEP4 gene, and proteinase B, the product of the PRB1 gene.
  • protease- deficient strains Another known example is the use of protease- deficient strains.
  • Some vacuolar protease- deficient strains of Pichia are already available, such as Pichia methanolica strain disclosed in WO 99/14347 (Zymogenetics, Inc.), which is deficient in proteinase A or proteinase B activity, or Pichia pastoris strains SMD1 168, SMD1163 or SMD1 165, which posses a substantial decrease in or elimination of proteinase A, carboxypeptidase Y and/or proteinase B activities.
  • it is not easy to create protease-deficient strains Indeed the proteolytic processes in eukaryotic organisms are quite complicated and involved in cell metabolism.
  • Pichia pastoris remains unsolved. Surprisingly the Applicant was able to overcome this problem in the present invention.
  • the present invention provides a Pichia pastoris cell suitable for the production of heterologous polypeptides, characterized in that said cell has been modified by recombinant DNA technology so that the gene for endogenous subtilisin Sub2 encoded by the DNA sequence comprising SEQ ID NO: 1 or variants or fragments thereof is completely or partially inactivated.
  • the invention further provides a method of producing a heterologous polypeptide in the Pichia pastoris cell according to the invention, said method comprising: (a) introducing into said cell a nucleic acid sequence encoding for a polypeptide;
  • step (b) cultivating the cell of step (a) in a culture medium under conditions
  • the invention also provides method for improving the production of heterologous polypeptides by a Pichia pastoris cell comprising inactivating completely or partially the gene for endogenous subtilisin Sub2 encoded by the DNA sequence comprising SEQ ID NO: 1 or variants or fragments thereof in said Pichia pastoris cell.
  • Another aspect of the invention is a method for obtaining the Pichia pastoris cell of the invention, wherein the inactivation of the subtilisin Sub2 gene is obtained by:
  • subtilisin Sub2 a DNA construct comprising the subtilisin Sub2 gene, wherein a part of said gene has been substituted, deleted, or extra DNA has been inserted within said subtilisin Sub2 gene
  • FIG. 1 Further aspect of the invention is a method for obtaining the Pichia pastoris cell of the invention, wherein the inactivation of the subtilisin Sub2 gene is obtained by using anti- sense technology, said method comprising:
  • FIG. 1 Overexpression of P. pastoris SUB I and SUB2 genes in P. pastoris KM71. Sub2 is over produced in P. pastoris KM71 -Si752
  • Figure 3 Use of GS l ⁇ 5-sub2 for heterologous protease production, (lanes 2 and 5), and SUB2 disruption in P. pastoris strains producing secreted proteases (lanes 4 and 7).
  • Figure 5 Generic plasmid for heterologous polypeptide expression in Pichia pastoris.
  • FIG. 6 Chromosomal integration of pKS l in Pichia pastoris GS 1 15 to generate GS l l 5-sub2 strain.
  • SUB2 Part of SUB2;
  • BLE S. hindustanus BLE gene for resistance to zeocin.
  • Plasmid pKS2 sequence is 100% identical to GS 1 ⁇ 5-sub2 DNA.
  • Pichia pastoris The methylotrophic yeast Pichia pastoris has been used successfully to express a wide range of heterologous proteins; one of the main interests being that Pichia pastoris secretes low levels of native proteins. Therefore it is well known that Pichia pastoris is a powerful tool to individually produce numerous secreted heterologous proteases as recombinant proteins in substantial amounts, without endogenous secreted proteases
  • proteases contaminating proteases. This is very useful when highly purified proteases are needed to analyze for example different steps in protein and peptide digestion.
  • the availability of recombinant proteases assisted many studies in the fields of secreted protease biochemistry, crystallography and clinical microbiology. Using a reverse genetic approach (from gene to protein), it was also possible to synthesize proteases which were revealed by genome sequencing, but which were not yet discovered and remained so far putative.
  • the Applicant used Pichia pastoris to produce recombinant leucine aminopeptidases, dipeptidyl-pepptidases and trieptidyl-pepptidases of the sedolisin family. Surprisingly and contrary to what has been known and expected, the Applicant realized that purified fractions of the above-mentioned proteases contained endoproteolytic activity. Thus in the present invention, the Applicant identifies and characterizes a contaminating protease of the subtilisin family which is secreted in minor amount by Pichia pastotris during growth in methanol medium.
  • Pichia pastoris genome harbours two genes encoding serine proteases of the subtilisin family (Sub): Subl codes for the vacuolar Proteinase B which is not secreted in culture medium, whereas Sub2 codes for a subtilisin which is secreted in low amount (3ng/ml culture medium of cells at OD 50 in methanol medium).
  • proteolytic activity from Pichia pastoris GS1 15 and KM71 has been studied. Two distinct gelatinolytic activities in Pichia pastoris cell culture supernatant were detected in SDS-PAGE gels containing gelatin as a substrate. One protease (proteolytic component) showed a distinct band and had an apparent molecular mass of 40 kDa
  • the Applicant searched to disrupt the gene encoding vacuolar protease B (Prb) in GS 1 15 strain.
  • Protease B is a serine protease of the subtilisin family and was postulated to be secreted by Pichia pastoris.
  • a gene encoding the putative Pichia pastoris Prb (ppPRB) was cloned from a Pichia pastoris genomic library as described in material and methods using Saccharomyces cerevisiae PRB DNA as a probe. ppPRB targeted disruption did not allow to abolish gelatinolytic activity in Pichia pastoris cell culture supernatant. Therefore the Applicant searched to clone another gene encoding a subtilisin for subsequent disruption.
  • Pichia pastoris transformants producing various heterologous proteases were found to secrete more Sub2 than GS 115 or KM71. This observation contrasts with previous findings made in niger, Trichoderma reesei and S. cerevisiae. Endoplasmic reticulum (ER)-associated stress generated by expression of heterologous secreted proteins or polypeptides was found to lead to the transcriptional down-regulation of some genes encoding secreted proteins, an effect termed repression under ER stress (RESS) which may be independent of the UPR.
  • ER Endoplasmic reticulum
  • the present invention provides a Pichia pastoris cell suitable for the production of heterologous polypeptides, characterized in that said cell has been modified by recombinant DNA technology so that the gene for endogenous subtilisin Sub2 encoded by the DNA sequence comprising SEQ ID NO: 1 or variants or fragments thereof is completely or partially inactivated.
  • Pichia pastoris cell of the invention is derived from any Pichia pastoris cell conventionally used for the heterologous expression of polypeptides or proteins.
  • the Pichia pastoris cell of the invention is derived from the parental cell selected from the group comprising wild type Pichia pastoris cell, Pichia pastoris GSl 15 strain, Pichia pastoris SMD1168 and SMD1168H strains, Pichia pastoris KM71 strain, Pichia pastoris SMD 1163 strain or Pichia pastoris SMD1 165 strain.
  • the recombinant DNA technology is specific or random mutagenesis or specific deletion, insertion and/or substitution in DNA sequence encoding the gene for endogenous subtilisin Sub2, gene disruption or gene replacement techniques, anti-sense techniques, or a combination thereof.
  • the Pichia pastoris cell of the present invention can further comprise a DNA sequence encoding for a heterologous polypeptide of interest.
  • the heterologous polypeptide is selected from the group consisting of therapeutic proteins, antibodies or enzymes.
  • subtilisin Sub2 protease is a serine protease having the activity at pH 6 - 9.5.
  • the subtilisin Sub2 protease is a Pichia pastoris subtilisin Sub2 protease (Sub2) encoded by a cDNA sequence comprising SEQ. ID. No. l , or variants, or fragments thereof or a sequence homologous thereto.
  • DNA is intended to include nucleic acid molecules or DNA molecules (such as cDNA or genomic DNA), RNA molecules (such as mRNA), analogs of the DNA or RNA generated using nucleotide analogs, derivatives, variants fragments and homologs thereof.
  • the nucleic acid molecule may be single- stranded or double- stranded.
  • DNA sequence homology is determined as the degree of identity between the two sequences indicating a derivation of the first sequence from the second.
  • Identity as known in the art and used herein, is a relationship between two or more nucleic acid sequences, as determined by comparing the sequences. It also refers to the degree of sequence relatedness between nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. Identity and similarity are well known terms to one skilled in the art and they can be calculated by conventional methods (for example see Computational Molecular Biology, Lesk, A. M. ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W.
  • the homologous DNA sequence exhibits a degree of identity preferably of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% with SEQ ID NO: l ; more preferably 90% identity with SEQ ID NO: l ; and most preferably at least 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1.
  • the invention also includes variant DNA sequences of SEQ ID NO: 1 , which still encode a protease that maintains Sub2 biological activity, or a fragment of SEQ ID NO: 1.
  • variants or “variants of a sequence” is meant a nucleic acid sequence or DNA sequence that vary form the reference sequence by conservative nucleic acid
  • variants encompass as well degenerated sequences, sequences with deletions and insertions, as long as such modified sequences exhibit the same biological activity as the reference sequence (i.e. encoding a protease having Sub2 biological activity).
  • the invention further includes DNA sequence whose sequence is complementary to SEQ ID NO: 1 , including DNA fragments that are complementary to SEQ ID NO: l as well.
  • the complementary DNA sequence is sufficiently complementary to SEQ ID NO: l that it can bind (via hydrogen bond) with little or no mismatches to SEQ ID NO: l , thereby forming a stable duplex.
  • the term "complementary” refers to Watson-Crick or Hoogsteen base pairing between nucleotide units of a nucleic acid molecule.
  • Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice.
  • a DNA or nucleic acid fragment encoding a biologically-active fragment of Sub2 protease can be prepared by isolating a fragment SEQ ID NO: 1 that encodes a protease having a biological activity of Sub2 protease of the invention (the biological activity of Sub2 protease of the invention is described above), expressing the encoded portion of Sub2 protease (for example, by recombinant expression in vitro) and assessing the activity of the encoded fragment of protease.
  • biological activity refers to the natural or normal function of Sub2 protease of the invention, for example the ability to degrade proteins.
  • Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution.
  • Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differ from it with respect to certain components or side chains. Analogs may be synthetic or from a different evolutionary origin and may have a similar or identical biological activity compared to wild type.
  • Homologs or orthologs are nucleic acid sequences or amino acid sequences of a particular gene that are derived from different species.
  • Derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described herein.
  • Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention as described herein.
  • DNA sequences encoding Sub2 protease of the invention from other species, and, thus, that have a DNA sequence that differs from SEQ ID NO: 1 are intended to be within the scope of the invention.
  • DNA molecules corresponding to natural allelic variants and homologues of Sub2 protease cDNAs of the invention can be isolated based on their homology to Sub2 protease DNA disclosed herein using the fungal cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • the subtilisin Sub2 protease is a Pichia pastoris subtilisin Sub2 protease (Sub2) having the amino acid sequence comprising the sequence SEQ ID NO: 2, or a sequence homologous thereto.
  • Amino acid sequence homology as used herein is similarly determined as the degree of identity between two sequences indicating a derivation of the first sequence from the second.
  • the present invention is also directed to variants of subtilisin Sub2 protease.
  • variant refers to a polypeptide or protein having an amino acid sequence that differ to some extent from a native SEQ. ID. NO: 2, which is an amino acid sequence that vary from the native sequence by conservative amino acid substitutions, whereby one or more amino acids are substituted by another with same characteristics and conformational roles.
  • the amino acid sequence variants possess substitutions, deletions, side-chain modifications and/or insertions at certain positions within the amino acid sequence of the native amino acid sequence.
  • Conservative amino acid substitutions are herein defined as exchanges within one of the following five groups: I. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, Gly
  • Pichia pastoris DNAs encoding P. pastoris PrB (Sub l) and Sub2 were independently cloned in Pichia pastoris expression vectors and overexpressed in Pichia pastoris.
  • the corresponding culture medium of Pichia pastoris grown in methanol inducing medium contained about 10 ⁇ g ml "1 of 40 kDa Sub2 which could be detected on SDS-PAGE gels stained with Coomassie brilliant blue (Fig. 2, lane 4).
  • Sub2 produced by gene overexpression had the same electrophoretic mobility than the enzyme secreted by GS 1 15 strain (Fig. 4).
  • the enzyme was active in Tris-HCl buffer between pH 7.0 and 9.5 on resorufm-labeled casein and keratine azure with a broad peak of optimum activity between pH 7.5 and 9.0.
  • Sub2 preproprotein is encoded by 1431 nucleotides starting from the ATG codon (477 aa). Based on alignment with different fungal subtilisins such as Aspergillus fumigatus ALP1 (Jaton-Ogay et al., 1992), the N-terminal aa sequence of the mature Sub2 is apparently preceded by a polypeptide of 179 aa, and the mature protein is 308 aa long. Inspection of the N- terminal aa sequence of the precursor beginning from the Metl residue suggested the existence of a signal peptide in the protease precursor polypeptide with an hydrophobic core.
  • the mature protein generated after cleavage of the pre and pro sequences from the 308 aa precursor have a calculated molecular mass of 34 kDa.
  • the primary structure of the mature protein contains four N-linked glycosylation sequences Asn-X- Ser, However on SDS-PAGE gels, the protein is not reduced by endo-H treatment indicating that the enzyme is not glycosylated.
  • Pichia pastoris cell modification
  • the Pichia pastoris cell is modified by recombinant DNA technology known to the person skilled in the art.
  • Said recombinant DNA technology is, but not limited to, specific or random mutagenesis or specific deletion, insertion and/or substitution in DNA sequence SEQ ID NO: 1 encoding the gene for endogenous subtilisin Sub2, gene disruption or gene replacement techniques, anti-sense techniques, or a combination thereof.
  • the gene sequences (DNA sequences) respectively responsible for production of subtilsin Sub2 protease activity may be inactivated or partially or entirely eliminated.
  • a Pichia pastoris cell of the invention expresses reduced or undetectable levels of subtilisin Sub2 protease or expresses functionally inactive subtilisin Sub2 protease.
  • the said inactivation is obtained by modification of the respective structural or regulatory regions encoded within the subtilisin Sub2 protease genes of interest.
  • Mutagenesis may be performed using a suitable physical or chemical mutagenising agent.
  • a physical or chemical mutagenising agent suitable for the present purpose include, but are not limited to, ultraviolet (UV) irradiation, hydroxylamine, N- methyl-N'-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulfite, formic acid, and nucleotide analogues.
  • the mutagenesis is typically performed by incubating the cell to be mutagenised in the presence of the mutagenising agent of choice under suitable conditions, and selecting for cells showing a significantly reduced production of subtilisin Sub2.
  • Modification may also be accomplished by specific deletion, insertion and/or substitution in DNA sequence SEQ ID NO: 1 encoding the gene for endogenous subtilisin Sub2. This can be achieved by removal, introduction and/or substitution of one or more nucleotides in the structural sequence or a regulatory element required for the transcription or translation of the structural sequence.
  • nucleotides may be inserted or removed so as to result in the introduction of a stop codon, the removal of the start codon or a change of the open reading frame of the structural sequence.
  • the modification or inactivation of the structural sequence or a regulatory element thereof may be accomplished by site-directed mutagenesis or PCR generated mutagenesis in accordance with methods known in the art.
  • the present invention provides a method for obtaining the Pichia pastoris cell of the invention, wherein the inactivation of the subtilisin Sub2 gene is obtained by:
  • subtilisin Sub2 a DNA construct comprising the subtilisin Sub2 gene, wherein a part of said gene has been substituted, deleted, or extra DNA has been inserted within said subtilisin Sub2 gene
  • the modification may be performed in vivo, i.e. directly on the cell expressing the subtilisin Sub2 protease genes, it is presently preferred that the modification be performed in vitro as exemplified below.
  • a convenient way to inactivate or reduce the subtilisin Sub2 protease production in a Pichia pastoris cell is based on techniques of gene interruption.
  • a DNA sequence corresponding to the endogenous gene or gene fragment of interest is mutagenised in vitro. Said DNA sequence thus encodes a defective gene which is then transformed into the host cell. By homologous recombination, the defective gene replaces the endogenous gene or gene fragment. It may be desirable that the defective gene or gene fragment also encodes a marker which may be used to select for transformants in which the respective gene encoding subtilisin Sub2 protease has been modified or destroyed.
  • the modification or inactivation of the DNA sequence may be performed by established anti-sense techniques using a nucleotide sequence complementary to an encoding sequence for subtilisin Sub2, e.g. the nucleotide sequences presented as SEQ. ID. No. l .
  • the anti-sense technology and its application is described in detail in US Patent No. 5, 190,931 (University of New York).
  • the present invention provides also a method for obtaining the Pichia pastoris cell of the invention, wherein the inactivation of the subtilisin Sub2 gene is obtained by using anti- sense technology, said method comprising:
  • the Pichia pastoris host cell of the invention does not express subtilisin Sub2 protease or expresses significantly reduced levels of subtilisin Sub2 protease activity.
  • the level of this proteolytic activity expressed by the Pichia pastoris host cell is individually reduced more than about 50%, preferably more than about 85%, more preferably more than about 90%, and most preferably more than about 95%.
  • this proteolytic activity in the Pichia pastoris host cell of the invention may be reduced in any combination.
  • the product expressed by the Pichia pastoris host cell is essentially free from proteolytic activity due to subtilisin Sub2 protease. Production of heterologous polypeptides
  • subtilisin Sub2 protease By the method of the invention, the proteolytic activity of subtilisin Sub2 protease is eliminated or significantly reduced, thereby improving the stability, purity and increasing the yield of susceptible polypeptides or proteins synthesized by the Pichia pastoris host cell of the invention.
  • the invention also provides a method of producing a heterologous polypeptide in the Pichia pastoris cell according to the invention, said method comprising:
  • step (b) cultivating the cell of step (a) in a culture medium under conditions
  • Another aspect of the invention provides also a method for improving the production of heterologous polypeptides by a Pichia pastoris cell comprising inactivating completely or partially the gene for endogenous subtilisin Sub2 encoded by the DNA sequence comprising SEQ ID NO: 1 in said Pichia pastoris cell. Improving the production of heterologous polypeptides refers not only to increasing the yield of production of heterologous polypeptides of interest, but also to obtaining pure heterologous polypeptides, such as proteases, in the culture medium without a contaminating endogenous protease secreted by Pichia pastoris. This serves as a first step in purification of the expressed heterologous polypeptides.
  • the desired end product i.e. the heterologous polypeptide or protein expressed by the Pichia pastoris cell of the invention, may be any polypeptides derived from prokaryotic sources, polypeptides derived from eukaryotic sources, and polypeptides derived from fungal sources other than the expression host.
  • a heterologous polypeptide is a protein or polypeptide gene product which is not native to the host cell, or is a native protein in which modifications have been made to alter the native sequence, or is a native protein whose expression is quantitatively altered as a result of a manipulation of a native regulatory sequence required for the expression of the native protein, such as a promoter, a ribosome binding site, etc., or other manipulation of the host cell by recombinant DNA techniques.
  • polypeptide and “protein” used interchangeably herein, are polymers of amino acids which are covalently linked through peptide bonds. These polymers of amino acids can be of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
  • the term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like.
  • a polypeptide can be a phosphopolypeptide, glycopolypeptide or metallopolypeptide.
  • heterologous polypeptides also include hybrid polypeptides which comprise a combination of partial or complete polypeptide sequences derived from at least two different polypeptides each of which may be homologous or heterologous with regard to the expression host.
  • heterologous polypeptides of the present invention also include: 1 ) naturally occurring allelic variations that may exist or occur in the sequence of polypeptides derived from the above prokaryotic, eukaryotic and final sources as well as those used to form the above hybrid polypeptides, and 2) engineered variations in the above heterologous polypeptides brought about, for example, by way of site specific mutagenesis wherein various deletions, insertions or substitutions of one or more of the amino acids in the heterologous polypeptides are produced.
  • Each of the above defined heterologous polypeptides is encoded by a heterologous DNA sequence which contains a stop signal which is recognized by the Pichia pastoris in which expression and secretion occurs. When recognized by those, the stop signal terminates translation of the mRNA encoding the heterologous polypeptide.
  • the heterologous polypeptide is selected from the group consisting of therapeutic proteins, antibodies and enzymes.
  • the enzyme is a proteolytic enzyme or protease.
  • the heterologous polypeptide is a hormone.
  • the heterologous protein expressed by the Pichia pastoris host cell may also be a precursor protein such as a zymogen, a hybrid protein, a protein obtained as a pro sequence or pre-pro sequence, or any other immature form.
  • Pichia pastoris cell of the invention may be accomplished by the person skilled in the art using standard recombinant DNA technology for the transformation or transfection of a host cell (vide, e.g., Sambrook et al, inter alia).
  • the Pichia pastoris cell of the invention can further comprise a DNA sequence encoding for a heterologous polypeptide of interest.
  • the Pichia pastoris cell is modified by methods known in the art for the introduction of an appropriate cloning vehicle, i.e. a plasmid or a vector, comprising a DNA sequence encoding for a heterologous polypeptide of interest.
  • the cloning vehicle may be introduced into the Pichia pastoris host cell either as an autonomously replicating plasmid or integrated into the chromosome.
  • the cloning vehicle comprises one or more structural regions operably linked to one or more appropriate regulatory regions.
  • the vector may be a plasmid or simply a linear DNA fragment. Once transformed into Pichia pastoris host, the vector may integrate into the genome.
  • plasmid and vector are sometimes used interchangeably as the plasmid is the most commonly used form of vector at present.
  • the invention is intended to include such other forms of expression vectors which serve equivalent functions and which are, or become, known in the art.
  • operably linked when describing the relationship between two DNA regions simply means that they are functionally related to each other.
  • a pre-sequence is operably linked to a peptide if it functions as a signal sequence, participating in the secretion of the mature form of the protein most probably involving cleavage of the signal sequence.
  • a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation.
  • the "structural regions” are regions of nucleotide sequences encoding the desired polypeptide.
  • the regulatory regions include promoter regions comprising transcription and translation control sequences, terminator regions comprising stop signals, and polyadenylation regions.
  • the promoter i.e. a nucleotide sequence exhibiting a transcriptional activity in the host cell of choice, may be one derived from a gene encoding an extracellular or an intracellular protein, preferably therapeutic proteins, antibodies or enzymes; the enzymes preferably being proteases.
  • the procedure consists of cloning the cDNA encoding the polypeptide of interest downstream of a signal sequence under the control of the AOXl promoter in a Pichia pastoris expression vector.
  • the Pichia pastoris acid phosphatase gene (PHOl) signal sequence or the a- factor signal peptide sequence are used for entering the secretory pathway of the yeast (Higgins and Cregg, 1998).
  • the construct which carries in addition to the cloned coding sequence of interest a gene for selection after transformation of Pichia pastoris is inserted into the Pichia pastoris genome at the AOXl locus via homologous recombination.
  • Selected transformants are screened for heterologous polypeptide production after induction of their encoding gene in a medium containing methanol.
  • the cloning vehicle may also include a selectable marker, such as a gene product which complements a defect in the host cell, or one which confers antibiotic resistance.
  • antibiotics useful as Pichia selection markers include hygromycin, phleomycin and basta.
  • Typical plasmid designed for heterologous polypeptide expression in Pichia pastoris is illustrated in the generic plasmid shown in Figure 5 (Higgins D.R., Current Protocols in Protein Science (1995)), wherein:
  • the 5'P ⁇ ox / is the promoter from the Pichia alcohol oxidase (AOXl) gene; it is used to drive methanol-inducible expression of the gene of interest.
  • the MCS is a multiple cloning site with unique restriction endonuclease sites; it is used to insert the gene of interest into the plasmid.
  • Transcription termination sequences are derived from the native Pichia AUX1 gene; they are used to promote efficient mRNA processing and polyadenylation.
  • HIS4 the wild-type gene for histidinol dehydrogenase, is a selectable marker; it is used to positively select for recombinant Pichia strains that have acquired the vector, which complements the auxotrophic his4 mutation.
  • the 3 ⁇ 1 sequence is derived from a region of the native gene that lies 3' to the transcription termination sequences; it is required for integration of vector sequence by gene replacement or gene insertion 3 ' to the chromosomal A 0X1 gene.
  • Sig is a DNA sequence juxtaposed between 5 'P AOXI and the MCS region; it encodes a protein secretion signal that is expressed as an N-terminal fusion to the protein of interest. This sequence directs the passage of the protein through the secretory pathway and targets proteins that carry it out of the cell.
  • Sig sequences that have been used for expression in Pichia pastoris include the S. cerevisiae mating type a prepro sequence (MF-la prepro), the Pichia acidphosphatase signal sequence (PHO), the S. cerevisiae invertase signal sequence, and several hybrid sequences.
  • the gene of interest may contain a sequence that encodes its own native secretion signal; that signal sequence may be functional in Pichia.
  • an r is a bacterial kanamycin resistance gene; it may be included to confer resistance to the drug G418.
  • Recombinant Pichia strains that contain multiple copies of Kan 1 are resistant to higher concentrations of G418 in a manner that correlates with the Kan r copy number.
  • the fl ori sequence is a bacterial single-strand origin of replication; this sequence can be included to generate single-stranded DNA in Escherichia coil to facilitate plasmid mutagenesis.
  • the present invention also encompasses simultaneously modifying Pichia pastoris cell (1) by recombinant DNA technologies (exemplified above) so that the gene for Sub2 is completely or partially inactivated and (2) by methods known in the art (exemplified above) for the introduction of an appropriate cloning vehicle, i.e. a plasmid or a vector, comprising a DNA sequence encoding for a heterologous polypeptide of interest.
  • an appropriate cloning vehicle i.e. a plasmid or a vector, comprising a DNA sequence encoding for a heterologous polypeptide of interest.
  • the culture medium conditions suitable for producing a heterologous polypeptide may be any conventional medium suitable for culturing the Pichia pastoris host cell of the invention known to the person skilled in the art, and formulated according to the principles of the prior art.
  • the medium preferably contains carbon and nitrogen sources as well as other inorganic salts.
  • Suitable media e.g. minimal or complex media, are available from commercial suppliers, or may be prepared according to published recipes, as in The Catalogue of Strains, published by The American Type Culture Collection. Rockville MD, USA. 1970.
  • the culture conditions, such as temperature, pH and the like, are those previously used with the Pichia pastoris host cell and will be apparent to the person skilled in the art.
  • the appropriate pH for fermentation is usually 6.0 - 8.0.
  • Pichia pastoris can be grown at 30°C in glycerol-based yeast media [0.1 M potassium phosphate buffer at pH 6.0, containing 10 g/L yeast extract, 20 g/L peptone, 13 g/L yeast nitrogen base without amino acids (Becton-Dickinson, Sparks, MD), 10 ml/L glycerol and 40 mg/L biotin].
  • Pichia pastoris cells are then harvested and resuspended in the same culture medium with methanol instead of glycerol and incubated for few more days, usually 2 days. Then, the culture supernatant is harvested after centrifugation at for example 3000 x g, 4°C, 5 min.
  • the desired protein is recovered by conventional methods of protein isolation and purification from a culture medium.
  • Well established purification procedures include separating the cells from the medium by centrifugation or filtration, precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, and chromatographic methods such as ion exchange chromatography, gel filtration chromatography, affinity chromatography, and the like.
  • Pichia pastoris GS 115 and KM71 (Invitrogen) and plasmid pPICZA (Invitrogen) were used.
  • Two primers PI (5 '-GGYCACGGIACICACGTIGCIGGIAC-3 '; I is inosine) (SEQ ID NO: 5) and P2 (5 '-GTGRGGIGTIGCCATIGAIGTICC-3 ' ; I is inosine) (SEQ ID NO : 6) were designed from alignment of genes encoding fungal subtilisins. Their nucleotide sequence encoded highly conserved amino acid sequences in subtilisin.
  • pKS l The resulting plasmid (pKS l) (Fig.6) corresponded to plasmid pPICZA in which the AOX1 promotor was replaced by part of the putative second Pichia pastoris subtilisin.
  • pKS l was cut by a Bsu961 (Saul) unique restriction site in the subtilisin DNA cloned fragment, and plasmidic linearized DNA was used to transform Pichia pastoris GS 1 15 and KM71. Transformants were selected on YPD to which was added zeocin at a concentration of 100 ⁇ g/ml and subsequently screened for gelatinolytic activities on SDS-PAGE.
  • One GS 1 15 transformant among 10 showed no proteolytic component at 40kDa. This clone was called GS 1 ⁇ 5-sub2.
  • Pichia pastoris GS 1 15-sub2 strain was deposited on 4 December 2009 and has accession number IHEM 23445.
  • PCR product was purified using a PCR purification kit (Roche Diagnostics), digested by restriction enzymes Sful and Xhol for which a recognition site was previously designed at the 5 ' extremity of the primers, and ligated to pPICZA cut with the same restriction enzymes Sful and Xhol.
  • the generated plasmid was called pKS 129. P.
  • pastoris KM71 was transformed by electroporation with 10 ⁇ g of p S129 linearized by Dral as previously described. Transformants were selected on YPD to which was added zeocin at a concentration of 100 ⁇ g/ml and subsequently screened for Sub2 overproduction in culture supernatant. One clone among 10 showed was found to secrete Sub2 at a rate of 10 ⁇ g/ml. This clone was called
  • biotin, 5 x 10 % amino acids ⁇ i.e. 5 x 10 % (w/v) of each L-glutamic acid, L- methionine, L-lysine, L-leucine, L-iso leucine)] were screened for insertion of the construct at the AOX1 site on minimal methanol plates [ 1.34% (w/v) YNB without amino acids, 4 x 10 % (w/v) biotin, 0.5% (v/v) methanol)].
  • Transformants unable to grow on media containing only methanol as a carbon source were assumed to contain the construct at the correct yeast genomic location by integration events in the AOX1 locus displacing the AOX1 coding region.
  • the selected transformants were grown to near saturation (OD 20 at 600 nm) at 30°C in 10 ml of glycerol-based yeast media (0.1 M potassium phosphate buffer at pH 6.0, containing 1 % (w/v) yeast extract, 2% (w/v) peptone, 1.34% (w/v) YNB without amino acids, 1% (v/v) glycerol and 4 x 10 % (w/v) biotin). Cells were harvested and resuspended in 2 ml of the same medium with 0.5% (v/v) methanol instead of glycerol and incubated for 2 days.
  • glycerol-based yeast media 0.1 M potassium phosphate buffer at pH 6.0, containing 1 % (w/v) yeast extract, 2% (w/v) peptone, 1.34% (w/v) YNB without amino acids, 1% (v/v) glycerol and 4 x 10 % (w/v) biotin).
  • Resorufin-labeled casein substrate was not sensitive enough to detect proteolytic activity in Pichia pastoris culture supernatants. Therefore, proteolytic activity of GS 1 15 and M71 culture supernatants were compared with serial dilution of purified recombinant Sub2 extract to assess the amount of protease secreted by the yeast (Fig 4). One ml of culture supernatant was found to contain approximately 3 ng of Sub2. Increased activities were found in Pichia pastoris transformants producing various recombinant proteases (AfuDppIV and AfuLapl). Pichia pastoris PRB gene cloning
  • Pichia pastoris ⁇ genomic was constructed as previously described for Candida parapsilosis (de Viragh). Recombinant plaques (2 x 10 4 ) of the genomic library were immobilized on GeneScreen nylon membranes (NEN Life Science products). The filters were hybridized with 2 P-labelled Saccharomyces cerevisiae PRB DNA probe under low-stringency conditions (Monod et al., 1994). All positive plaques were purified and the associated bacteriophage DNAs were isolated as described previously (Grossberger, 1987).
  • Extracts were analyzed by SDS-PAGE (Laemmli, 1970) with a separation gel of 12% polyacrylamide.
  • gelatin at a concentration of 0.1% was added in the gel.
  • 10 ⁇ of P. pastoris culture supernatant were treated with an equal volume of SDS-PAGE sample buffer without beta-mercapthoethanol (125 mM Tris, pH 6.8, 4% SDS, 20% glycerol, 0.002

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Abstract

L'invention concerne des micro-organismes et des procédés de production de protéines. L'invention se rapporte, en particulier, à une cellule hôte destinée à l'expression de protéines hétérozygotes, laquelle cellule hôte, Pichia pastoris, a été génétiquement modifiée de manière que le gène de la protéase endogène sécrétée subtilisine SUB2 a été complètement ou partiellement inactivé.
PCT/IB2010/055777 2009-12-14 2010-12-13 Pichia pastoris déficiente en protéase endogène sécrétée WO2011073883A1 (fr)

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